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Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides.

Lau JP, Weatherdon KL, Skalski V, Hedley DW - Br. J. Cancer (2004)

Bottom Line: To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity.Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity.While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9.

ABSTRACT
Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.

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Related in: MedlinePlus

Endonuclease activity of MiaPaCa and Panc-1 cells treated with antisense oligonucleotides. The MiaPaCa and Panc-1 cell lines were exposed to antisense or sense oligonucleotides for various time points. Cell lysates were collected and analysed using the endonuclease assay. C – control; AS – antisense; S – sense.
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fig5: Endonuclease activity of MiaPaCa and Panc-1 cells treated with antisense oligonucleotides. The MiaPaCa and Panc-1 cell lines were exposed to antisense or sense oligonucleotides for various time points. Cell lysates were collected and analysed using the endonuclease assay. C – control; AS – antisense; S – sense.

Mentions: Both cell lines were transfected with antisense or sense oligonucleotides and APE/ref-1 protein levels were analysed 12, 24, 48 and 72 h post-transfection. Using both Western blot and flow cytometry, there were statistically significant decreases in protein levels detected after 24 h of transfection with MiaPaCa-2 decreasing three-fold (P<0.001) and Panc-1 decreasing four-fold (P<0.001). After 48 h post-transfection, the APE/ref-1 levels had substantially recovered. The level of endonuclease activity was assessed in cell lysates, obtained from the same flasks as the samples taken for protein measurements. As illustrated in Figure 5Figure 5


Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides.

Lau JP, Weatherdon KL, Skalski V, Hedley DW - Br. J. Cancer (2004)

Endonuclease activity of MiaPaCa and Panc-1 cells treated with antisense oligonucleotides. The MiaPaCa and Panc-1 cell lines were exposed to antisense or sense oligonucleotides for various time points. Cell lysates were collected and analysed using the endonuclease assay. C – control; AS – antisense; S – sense.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747714&req=5

fig5: Endonuclease activity of MiaPaCa and Panc-1 cells treated with antisense oligonucleotides. The MiaPaCa and Panc-1 cell lines were exposed to antisense or sense oligonucleotides for various time points. Cell lysates were collected and analysed using the endonuclease assay. C – control; AS – antisense; S – sense.
Mentions: Both cell lines were transfected with antisense or sense oligonucleotides and APE/ref-1 protein levels were analysed 12, 24, 48 and 72 h post-transfection. Using both Western blot and flow cytometry, there were statistically significant decreases in protein levels detected after 24 h of transfection with MiaPaCa-2 decreasing three-fold (P<0.001) and Panc-1 decreasing four-fold (P<0.001). After 48 h post-transfection, the APE/ref-1 levels had substantially recovered. The level of endonuclease activity was assessed in cell lysates, obtained from the same flasks as the samples taken for protein measurements. As illustrated in Figure 5Figure 5

Bottom Line: To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity.Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity.While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9.

ABSTRACT
Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.

Show MeSH
Related in: MedlinePlus